| ObjectiveChondroitin sulfate (CS) ubiquitously distributes on cell surfaces and in the extracellular matrix (ECM) of mammalian animals, and is particularly abundant in bones, tendons, blood vessels, nerve tissues, and cartilage. CS, a sulfated glycosaminoglycan (GAG), is composed of repeating disaccharide units of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), which is commonly sulfated at the C-4and/or C-6of GalNAc in mammals. CS has been discovered to have various biological functions including anti-oxidation, anti-inflammation, anti-atherosclerosis, immunoregulation, neuroprotection, regulating cell adhesion and morphogenesis, and low or no toxicity with long term administration. The market for products containing CS and glucosamine is developing in North America, Europe, South East Asia and Australia. In United States, CS is recognized as a "dietary supplement". While in Europe, CS is marketed as symptomatic slow-acting drugs for osteoarthritis and is widely used for the relief of symptoms and pain of arthritic diseases. However, its clinical use has encountered certain limitations because of its poor intestinal absorption, resulting from its high molecular weight, charge density, as well as hydrophilicity. Therefore, intestinal absorption become one of critical factors for the successful application of CS and CS derivatives in the treatment of osteoarthritis and atherosclerosis. In order to get around this drawback, researchers have tried many strategies including adding absorption enhancers, preparing bioadhesive nanoparticles or prodrugs, as well as increasing drug lipophilicity. In this work, we prepared a new amphiphilic CS, a-linolenic acid (a-LNA)-low molecular weight CS (LMCS) conjugate (a-LNA-LMCS), which can form stable nanomicelles in aqueous media. The oral bioavailability of a-LNA-LMCS micelles was evaluated in vivo and in vitro, and the possible transport mechanism of a-LNA-LMCS micelles was determined by using Caco-2cell monolayers. Furthermore, the effect and possible mechanisms of anti-atherosclerosis of CS and its derivative were investigated by using apolipoprotein E-deficient(ApoE-/-) mice.Method1. Preparation and characterization of a-LNA-LMCSLMCS was prepared by using a controlled oxidative depolymerization process in the presence of hydrogen peroxide. Then, LMCS was hydrophobically modified with a-linolenic acid (a-LNA) to obtain a series of amphiphilic CS (a-LNA-LMCSs) with different degree of a-LNA substitution. Structural characterizations of a-LNA-LMCSs were analized by FTIR,1HNMR, TGA/DSC. The physicochemical properties of a-LNA-LMCSs in aqueous media were characterized by transmission electron microscopy (TEM), laser light scattering, zeta potential and fluorescence spectroscopy.2. In vitro and in vivo oral bioavailability and absorption mechanism study of CS and its derivativesThe oral bioavailability of a-LNA-LMCS micelles in vivo was evaluated by determining the a-LNA-LMCS concentrations in plasma levels following oral administration to rats in comparison with CS. Caco-2cell monolayers representing in vitro model of the intestinal epithelial barrier were used to determine the intestinal transport ability of a-LNA-LMCS micelles. Furthermore, confocal laser scanning microscope (CLSM) was used to study the transport mechanism of CS and its derivatives across the intestinal epithelial barrier.3. Anti-atherosclerosis effect and the mechanism study of LMCS and LNA-LMCS2 8-week-age ApoE-/-mice were randomly divided into8groups (13each), treated with atherogenic diet (15%fat and0.25%cholesterol) together with or without tested compounds. After16weeks of administration, mice were anesthetized with10%chloral hydrate, and then the blood samples were collected by cardiac puncture. The levels of triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in blood plasma were measured by an automatic biochemistry analyzer. The plasma levels of IL-6, TNF-a and C-reactive protein (CRP) were detected by enzyme-linked immuno sorbent assay (ELISA) kits. Meanwhile, part of the hearts and aortas were perfusion-fixed with4%paraformaldehyde for histological and morphological staining (Hematoxylin and eosin staining, Oil-Red-O staining, Masson staining) and immunohistochemistry staining (MAMO-2, a-SMA). The remaining part of the hearts and aortas were treated with PBS for real-time polymerase chain reaction (real-time PCR) and Western Blot. Real-time PCR was used to detect the mRNA levels of IL-6, TNF-a, CRP, MCP-1, VCAM-1and ICAM-1. Western blot was used to detect the protein expression levels of p-NF-κB, p-JNK, p-ERK1/2, MCP-1, VCAM-1and ICAM-1.Results1. Preparation and characterization of a-LNA-LMCSA series of α-LNA-LMCSs with different degree of a-LNA substitution were prepared and characterized. The results of FT-IR,1HNMR and TGA/DSC indicated that all samples had successfully undergone the esterification of the hydroxyl groups in the repeating disaccharide of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), which meant that the α-LNA was introduced as the side chains of LMCS. The degree of substitution (DS) of α-LNA-LMCSs ranged from0.034to0.123. TEM observation demonstrated that α-LNA-LMCS micelles were roughly smooth sphere morphology and had a narrow and unimodal size distribution. The mean diameters of α-LNA-LMCS micelles were in the range of78-117nm. The critical aggregation concentrations of α-LNA-LMCS micelles were in the range of0.016-0.20mg/mL. The zeta potential of a-LNA-LMCS micelles were in the range of-30～-20mV, and the high negative charge improved the stability of micelles in aqueous media.2. In vitro and in vivo oral bioavailability and absorption mechanism of CS and its derivativeThe absorptions of CS and its derivatives were evaluated in vivo. The maximum concentration (Cmax) of LNA-LMCS2was10.8±0.5mg/L at8.8±2.3h, which was obviously higher than that of CS and LMCS. LNA-LMCS2showed a much longer circulation time and its elimination t1/2was18.8±3.1h,1.7times and3.2times longer than that of CS and LMCS. The total body clearance (CL) of LNA-LMCS2was0.6±0.4L/(h-kg), which was5.6times and5.3times smaller than that of CS and LMCS, respectively. Moreover, the AUC0-24of LNA-LMCS2after intragastric administration was172.4±20.9mg/(L·h), extremely higher than that of CS,40.0±4.4mg/(L-h) and LMCS,55.2±4.4mg/(L·h), respectively (p<0.001). It was deduced that the oral bioavailability of CS has been significantly improved.Caco-2transport studies demonstrated that the apparent permeability coefficient (Papp) of LNA-LMCS2was significantly higher than that of CS and LMCS (p<0.001), and no significant effects on the overall integrity of the monolayer were observed during the transport process. In addition, the efflux ratios of a-LNA-LMCSs were remarkably lower than that of CS or LMCS (p<0.05), which indicated that a-LNA-LMCS micelles had inhibitory effect on P-glycoprotein (P-gp). The transepithelial electrical resistance (TEER) values of Caco-2cell monolayers incubated with a-LNA-LMCSs decreased significantly compared to that of the control groups incubated with CS and LMCS, indicating that a-LNA-LMCSs had some effects on the opening of intercellular tight junctions. Furthermore, evident alterations in the F-actin cytoskeleton were detected by CLSM observation following the treatment of the cell monolayers with a-LNA-LMCS micelles, which further certified the capacity of a-LNA-LMCS micelles to open the intercellular tight junctions rather than disrupt the overall integrity of the monolayer. Based on these results and the in vivo efficacy study, it was deduced that the modification of LMCS with a-LNA improved its intestinal absorption through the enhancement of the transcellular and paracellular transport across intestinal cells.3. Anti-atherosclerosis effect and the mechanism of LMCS and LNA-LMCS2ApoE-/-mice in8groups fed a atherogenic diet with or without LNA-LMCS2or LMCS did not differ in body weight (p>0.05), which indicated that these drugs had no effect on body weight of the mice. The levels of TC and LDL-C in the high-dose group of LNA-LMCS2or LMCS were significantly lower than that of control group (p<0.05). More importantly, the plasma levels of TNF-a, IL-6and CRP were significantly lower in the high dose group of LNA-LMCS2than those in model control group. These results suggest that LNA-LMCS2is effective in reducing lipid levels and mitigating the inflammatory response on atherosclerosis.By using en face analysis of the arteria aorta, lesion area was significantly smaller in the high dose groups of LNA-LMCS2and LMCS than in the control group (p<0.001and p<0.05, respectively). Atherogenesis level at the aortic sinus was evaluated by H&E and Oil-Red-O staining by ratio of total atherosclerotic lesion area to aortic valve ring area. The mean lesion size at the aortic sinus was smaller in the high dose groups of LNA-LMCS2and LMCS than in the control group (p<0.001and p<0.05, respectively). The result of Masson staining indicated that LNA-LMCS2and LMCS had little effect on the collagen fibers in plaques. Immunohistochemistry staining demonstrated that, compared with the control group, the numbers of macrophages in plaques were significantly less in the high dose groups of LNA-LMCS2and LMCS (p<0.05). At the same time, smooth muscle cells in plaques were significantly higher in the high dose groups of LNA-LMCS2and LMCS (p<0.05) compared than in the control group. All the results demonstrated that LNA-LMCS2and LMCS could decelerate the progression of atherosclerosis in ApoE-/-mice.The results of Western blot showed that LNA-LMCS2and LMCS could reduce the nuclear translocation of NF-κB by inhibiting the activity of ERK1/2, and further decrease the protein expressions of COX-2, MCP-1,VCAM-1and ICAM-1, which is consistent with the findings of RT-PCR. Based on these results, it was deduced that LNA-LMCS2and LMCS inhibited the atherosclerotic plaques through two ways:regulation of the lipid metabolism and anti-inflammation.Conclusions and significance1. A new series of amphiphilic polysaccharides, a-LNA-LMCSs, were successfully synthesized by using a-LNA as the hydrophobic chain. a-LNA-LMCS micelles were found to effectively increase the oral bioavailability of LMCS.2. The transport mechanism of LNA-LMCS2micelles across intestinal epithelial barrier via paracellular pathway and endocytosis was proved for the first time.3. High dose of LMCS and LNA-LMCS2(400mg/kg) can regulate the lipid metabolism and diminish the synthesis of pro-inflammatory enzymes and cytokines, which might be the mechanisms of anti-atherosclerosis effect of CS and its derivatives.4. This study has demonstrated that LNA-LMCS2is a new amphiphilic polysaccharide with good oral bioavailability and anti-atherosclerosis effect, and uncover the fundamental intestinal transport mechanisms and anti-atherosclerosis mechanisms of LNA-LMCS2. The results of this study will not only provide a good strategy to enhancing the intestinal absorption of polysaccharides, but also improve the development of CS based anti-atherosclerosis drug in the future. |
PDF Full Text Request